Orphanin-FQ/nociceptin (OFQ/N) modulates the activity of suprachiasmatic nucleus neurons. (1/646)

Neurons in the suprachiasmatic nucleus (SCN) constitute the principal circadian pacemaker of mammals. In situ hybridization studies revealed expression of orphanin-FQ/nociceptin (OFQ/N) receptor (NOR) mRNA in the SCN, whereas no expression of mRNA for preproOFQ/N (ppOFQ/N) was detected. The presence of OFQ/N peptide in the SCN was demonstrated by radioimmunoassay. SCN neurons (88%) responded dose-dependently to OFQ/N with an outward current (EC50 = 22.3 nM) that was reduced in amplitude by membrane hyperpolarization and reversed polarity near the theoretical potassium equilibrium potential. [Phe1psi(Ch2-NH)Gly2]OFQ/N(1-13)NH2 (3 microM), a putative NOR antagonist, activated a small outward current and significantly reduced the amplitude of the OFQ/N-stimulated current. OFQ/N reduced the NMDA receptor-mediated increase in intracellular Ca2+. When injected unilaterally into the SCN of Syrian hamsters housed in constant darkness, OFQ/N (1-50 pmol) failed to alter the timing of the hamsters' wheel-running activity. However, injection of OFQ/N (0.1-50 pmol) before a brief exposure to light during the midsubjective night significantly attenuated the light-induced phase advances of the activity rhythm. These data are consistent with the interpretation that OFQ/N acting at specific receptors modulates the activity of SCN neurons and, thereby, the response of the circadian clock to light.  (+info)

Cardiovascular effects of nociceptin in unanesthetized mice. (2/646)

We evaluated the systemic hemodynamic effects induced by nociceptin (NC) and NC-related peptides, including the NC receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2) in unanesthetized normotensive Swiss Morini mice. Bolus intravenous injection of NC decreased mean blood pressure and heart rate. The hypotensive response to 10 nmol/kg NC lasted <10 minutes, whereas a more prolonged hypotension was evoked by 100 nmol/kg (from 114+/-3 to 97+/-2 mm Hg at 10 minutes, P<0.01). The latter dose reduced heart rate from 542+/-43 to 479+/-31 beats/min (P<0.05) and increased aortic blood flow by 41+/-5% (P<0.05). Hypotension and bradycardia were also evoked by NC(1-17)NH2 and NC(1-13)NH2 fragments, whereas NC(1-13)OH and NC(1-9)NH2 were ineffective. Thiorphan, an inhibitor of neutral endopeptidase 24.11, enhanced the hypotension induced by NC(1-13)NH2 and revealed the ability of NC(1-13)OH to decrease mean blood pressure. [F/G]NC(1-13)NH2, a recently synthesized antagonist of the NC receptor, did not alter basal mean blood pressure or heart rate, but it prevented the hypotension, bradycardia, and increase in aortic blood flow evoked by NC. In contrast, [F/G]NC(1-13)NH2 did not alter the hypotension induced by bradykinin or endomorphin-1 (a micro-receptor agonist), and the bradycardia induced by leu-enkephalin (a delta-receptor agonist) or U504885 (a synthetic kappa-receptor agonist). In conclusion, NC and some of its fragments cause hypotension and bradycardia and increase aortic blood flow in mice, with the NC(1-13) sequence being critical for these biological effects. Our results also demonstrate that the compound [F/G]NC(1-13)NH2 is a potent and selective antagonist of the NC receptor in vivo.  (+info)

Central administration of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)-NH2 and orphanin FQ/nociceptin (OFQ/N) produce similar cardiovascular and renal responses in conscious rats. (3/646)

In vitro studies have shown that [Phe1Psi(CH2-NH)Gly2]OFQ/N(1-13)-NH2 (referred to as [FG]OFQ/N(1-13)-NH2) is the first selective antagonist to prevent the binding of the endogenous ligand orphanin FQ/Nociceptin (OFQ/N) at the orphan opioid-like receptor. In the present study, we examined the potential changes in cardiovascular and renal function produced by the i.c.v. injection of [FG]OFQ/N(1-13)-NH2 in conscious Sprague-Dawley rats. In conscious rats, i.c.v. injection of [FG]OFQ/N(1-13)-NH2 produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate (i.e., a water diuresis). The cardiovascular and renal excretory responses produced by i.c.v. [FG]OFQ/N(1-13)-NH2 were dose dependent and were similar in pattern but of longer duration than responses evoked by i.c.v. OFQ/N. In other animals, the i.c.v. injection of OFQ/N(1-13)-NH2, a potential metabolite of [FG]OFQ/N(1-13)-NH2, produced changes in cardiovascular and renal function that were comparable to those evoked by i.c.v. [FG]OFQ/N(1-13)-NH2. In contrast, OFQ/N(2-17), a fragment of OFQ/N [OFQ/N(1-17)], was inactive when administered centrally. Finally, studies were performed to determine whether [FG]OFQ/N(1-13)-NH2 may be an antagonist at the orphan opioid-like receptor receptor when administered centrally at a dose that alone was inactive. In these studies, i.c.v. pretreatment of animals with low-dose [FG]OFQ/N(1-13)-NH2 failed to prevent the cardiovascular and renal excretory response to i.c.v. OFQ/N. Although [FG]OFQ/N(1-13)-NH2 is reported to be an antagonist of the OFQ/N receptor in vitro, these findings indicate that this compound has agonist activity similar to that of the endogenous ligand OFQ/N when administered centrally in vivo.  (+info)

Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ. (4/646)

For the further elucidation of the central functions of nociceptin/orphanin FQ (noc/OFQ), the endogenous ligand of the G protein-coupled opioid receptor-like receptor ORL1, centrally acting specific antagonists will be most helpful. In this study it was found that the hexapeptide acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2), described in literature as partial agonist on ORL1 transfected in CHO cells, antagonizes the stimulation of [35S]-GTPgammaS binding to G proteins by noc/OFQ in membranes and sections of rat brain. The antagonism of the peptide was competitive, of high affinity (Schild constant 6.58 nM), and specific for noc/OFQ in that the stimulation of GTP binding by agonists for the mu-, delta-, and kappa-opioid receptor was not inhibited. The hexapeptide also fully inhibited the chronotropic effect of noc/OFQ on neonatal rat cardiomyocytes. It is suggested that Ac-RYYRIK-NH2 may provide a promising starting point for in vivo tests for antagonism of the action of noc/OFQ and for the further development of highly active and specific antagonists.  (+info)

Proteolytic degradation of hemoglobin by endogenous lysosomal proteases gives rise to bioactive peptides: hemorphins. (5/646)

Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin-7-related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.  (+info)

Pre- and postsynaptic actions of opioid and orphan opioid agonists in the rat arcuate nucleus and ventromedial hypothalamus in vitro. (6/646)

1. Using whole-cell patch clamp recording from neurones in an in vitro slice preparation, we have examined opioid- and orphanin FQ (OFQ)-mediated modulation of synaptic transmission in the rat arcuate nucleus and ventromedial hypothalamus (VMH). 2. Application of OFQ activated a Ba2+-sensitive and inwardly rectifying K+ conductance in approximately 50 % of arcuate nucleus neurones and approximately 95 % of VMH neurones. The OFQ-activated current was blocked by the nociceptin antagonist [Phe1Psi(CH2NH)Gly2]-nociceptin(1-13) NH2 (NCA), a peptide that on its own exhibited only weak agonist activity at high concentrations (> 1 microM). Similar current activation was observed with the mu agonist DAMGO but not delta (DPDPE) or kappa (U69593) agonists. 3. In arcuate nucleus neurones, DAMGO (1 microM), U69593 (1 microM) and OFQ (100 nM to 1 microM) but not DPDPE (1 microM) were found to depress the amplitude of electrically evoked glutamatergic postsynaptic currents (EPSCs) and decrease the magnitude of paired-pulse depression, indicating that opioid receptors were located presynaptically. 4. In VMH neurones, DAMGO strongly depressed the EPSC amplitude in all cells examined. DAMGO decreased the magnitude of paired-pulse depression, indicating that mu receptors were located presynaptically. U69593 weakly depressed the EPSC while OFQ and DPDPE had no effect. 5. In VMH neurones, DAMGO depressed the frequency of miniature EPSCs (-58 %) in the presence of tetrodotoxin and Cd2+ (100 microM), suggesting that the actions of mu receptors could be mediated by an inhibition of the synaptic vesicle release process downstream of Ca2+ entry. 6. The data presented show that presynaptic modulation of excitatory neurotransmission in the arcuate nucleus occurs through mu, kappa and the orphan opioid ORL-1 receptors while in the VMH presynaptic modulation only occurs through mu opioid receptors. Additionally, postsynaptic mu and ORL-1 receptors in both the arcuate nucleus and VMH modulate neuronal excitability through activation of a K+ conductance.  (+info)

Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors. (7/646)

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(CH2-NH)Gly2]Nociceptin(1-13)NH2 ([F/G]NC(1-13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1-13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHO(NCR)) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHO(NCR) cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1-13)NH2 was as potent as NC(1-13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHO(NCR) cells was NCNH2> or =NC=NC(1-13)NH2>NC(1-12)NH2> >NC(1-11)NH2. [F/G]NC(1-13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1-13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5+/-4.9% and 7.39, 58.9+/-6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1-13)NH2, displayed a small (instrinsic activity alpha = 0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1-13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1-13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.  (+info)

Further characterization of the ORL1 receptor-mediated inhibition of noradrenaline release in the mouse brain in vitro. (8/646)

Mouse brain slices preincubated with [3H]-noradrenaline or [3H]-serotonin were superfused with medium containing naloxone 10 microM; we studied whether nociceptin (the endogenous ligand at ORL1 receptors) affects monoamine release. Furthermore, the affinities of ORL1 ligands were determined using [3H]-nociceptin binding. The electrically (0.3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]-noradrenaline was inhibited by nociceptin and [Tyr14]-nociceptin (maximally by 80%; pEC50 7.52 and 8.28) but not affected by [des-Phe1]-nociceptin (pEC50<6). The ORL1 antagonist naloxone benzoylhydrazone antagonized the effect of nociceptin and [Tyr14]-nociceptin. The effect of nociceptin did not desensitize, was not affected by blockade of NO synthase, cyclooxygenase and P1-purinoceptors and was decreased by the alpha2-adrenoceptor agonist talipexole. Nociceptin also inhibited the evoked overflow in mouse cerebellar, hippocampal and hypothalamic slices in a manner sensitive to naloxone benzoylhydrazone. The electrically (3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]-serotonin was inhibited by nociceptin; naloxone benzoylhydrazone antagonized this effect. The affinities (pKi) for [3H]-nociceptin binding to mouse cortex membranes were: nociceptin, 8.71; [Tyr14]-nociceptin, 9.82; [des-Phe1]-nociceptin, <5.5; naloxone benzoylhydrazone, 5.85; naloxone, <4.5. In conclusion, nociceptin inhibits noradrenaline release in the mouse cortex via ORL1 receptors, which interact with presynaptic alpha2-autoreceptors on noradrenergic neurones. The effect of nociceptin does not desensitize nor does it involve NO, prostanoids or adenosine. Nociceptin also attenuates noradrenaline release from several subcortical regions and serotonin release from cortical slices by a naloxone benzoylhydrazone-sensitive mechanism.  (+info)